MiR-26a-5p as a useful therapeutic target for upper tract urothelial carcinoma by regulating WNT5A/β-catenin signaling

The role of miRNAs in cancer and their possible function as therapeutic agents are interesting and needed further investigation. The miR-26a-5p had been demonstrated as a tumor suppressor in various cancers. However, the importance of miR-26a-5p regulation in upper tract urothelial carcinoma (UTUC) remains unclear. Here, we aimed to explore the miR-26a-5p expression in UTUC tissues and to identify its regulatory targets and signal network involved in UTUC tumorigenesis. The miR-26a-5p expression was validated by quantitative real-time polymerase chain reaction (qPCR) using renal pelvis tissue samples from 22 patients who were diagnosed with UTUC and 64 cases of renal pelvis tissue microarray using in situ hybridization staining. BFTC-909 UTUC cells were used to examine the effects of miR-26a-5p genetic delivery on proliferation, migration and expression of epithelial-to-mesenchymal transition (EMT) markers. MiR-26a-5p was significantly down-regulated in UTUC tumors compared to adjacent normal tissue and was decreased with histological grades. Moreover, restoration of miR-26a-5p showed inhibition effects on proliferation and migration of BFTC-909 cells. In addition, miR-26a-5p delivery regulated the EMT marker expression and inhibited WNT5A/β-catenin signaling and expression of downstream molecules including NF-κB and MMP-9 in BFTC-909 cells. This study demonstrated that miR-26a-5p restoration may reverse EMT process and regulate WNT5A/β-catenin signaling in UTUC cells. Further studies warranted to explore the potential roles in biomarkers for diagnostics and prognosis, as well as novel therapeutics targets for UTUC treatment.

www.nature.com/scientificreports/ Epithelial-to-mesenchymal transition (EMT) is a process for the transformation of cancer cells from epithelial to mesenchymal types, which is clinically correlated with cancer metastasis and associated with recurrence and drug resistance in cancers 3,[12][13][14][15][16][17][18] . Thus, suppressing EMT process could inhibit the migration and invasion during cancer progression. Additionally, there was study showing that reduced miR-26a expression is associated with lung metastasis and poor overall survival of osteosarcoma patients 11 .
To date, whether miR-26a (miR-26a-5p) regulates EMT process of UTUC cells and the molecular mechanisms involved remain unclear and need further investigation. The aims of this study were first to validate the miR-26a-5p expression using UTUC samples. Subsequently, we investigated the tumor suppressor function and mechanism using a renal pelvis transitional cell carcinoma cell line (BFTC-909 cells) 3 .
Results miR-26a-5p expression was down-regulated in human UTUC tissues. According to our previous study 3 , several microRNAs were down-regulated in UTUC tissues compared with adjusted normal controls, including miR-26a-5p. In this study, we first validated the miR-26a-5p expression in UTUC samples using qPCR assay and tissue microarray (TMA) using in situ hybridization staining (ISH). As shown in Fig. 1A, miR-26a-5p was significantly down-regulated in UTUC samples (n = 22:14). Thereafter, we next assessed miR-26a-5p expression in renal pelvis transitional cell carcinoma TMA which contained 64 tissue sections. As shown in Fig. 1B, miR-26a-5p was expressed around cytoplasmic area and the ISH immunosignals decreased progressively in advanced tumor grades. The intensity score was significantly lower in grade 3 tumors than grade 1 tumors (Fig. 1C). Taken together, these data imply the tumor suppressor role of miR-26a-5p in UTUC tumorigenesis.
Restoration of miR-26a-5p reduced BFTC-909 cells proliferation and migration. According to the ISH data, we further investigate the effects of miR-26a-5p restoration on UTUC cell proliferation and migration. Thus, we transfected miR-26a-5p mimics into BFTC-909 cells which is a well-known in vitro model of UTUC 2,3 . The proliferation was exam by Water-soluble tetrazolium (WST-1) assay and migration ability was exam by trans-well assay, respectively. As showed in Fig. 2, the miR-26a-5p restoration significantly reduced cell number ( Fig. 2A) and migration ability (Fig. 2B,C). According to these results, we demonstrated that miR-26a-5p could serve as a tumor repressor in UTUC cell growth and migration. www.nature.com/scientificreports/ Restoration of miR-26a-5p inhibited EMT processes. Epithelial mesenchymal transition processes has been implicated in carcinogenesis and confers metastatic properties 12 . Thus, we analyzed the effect of miR-26a-5p restoration in the EMT processes in UTUC while the E-cadherin, vimentin, α-SMA and fibronectin expression levels were assayed. As showed in Fig. 3, miR-26a-5p significantly restored cellular epithelial E-cadherin expression and inhibited α-SMA, fibronectin and vimentin expression. Moreover, the qPCR and western blot analyses similarly showed miR-26a-5p restoration significantly enhanced E-cadherin expression in mRNA and protein levels. Conversely, miR-26a-5p restoration reduced α-SMA, fibronectin and vimentin expression (Fig. 4). Together, these results indicated that miR-26a-5p might prohibit UTUC metastasis by regulating the EMT processes in UTUC cells. www.nature.com/scientificreports/ miR-26a-5p overexpression inactivation of WNT5A/β-catenin signaling in UTUC cells. The oncogenic mechanism underlying miR-26a-5p remains unclear. Because WNT5A/β-catenin signaling has been delineated to participate in the signal transduction of miR-26a-5p 6,19,20 , we compared the expression profiles of WNT5A/β-catenin signaling mediators between the UTUC cells with and without miR-26a-5p gene delivery. By using immunofluorescence staining, the data showed that miR-26a-5p restoration down-regulated the β-catenin and WNT5A expression (Fig. 5A,B) in UTUC cells. Furthermore, qPCR and immunoblot analyses confirmed that miR-26a-5p restoration reduced the mRNA and protein levels of β-catenin and WNT5A expression in UTUC cells (Fig. 6).

Discussion
Aberrant miRNA expression have been regarded a hallmark in all cancers, which disrupts the normal function of their targets and leads to the validation of tumor phenotypic transformation and metastasis as well as to drug resistance 22 . Thus, to fine out the gene regulatory effects of miRNAs may help exploit the potential treatment strategy in cancer diseases. There are studies suggesting that suppression of oncogenic miRNAs could become a reliable tool for improving the cancer therapy 3,8,22 . www.nature.com/scientificreports/ miR-26a has been reported to act as tumor suppressor via targeting specific downstream genes in several human cancers, such as melanoma, thyroid, prostate cancer, laryngeal squamous cell and hepatocellular carcinoma 6,23-29 . However, the role of miR-26a-5p in UTUC tumorigenesis was largely unknown. In the present study, we report for the first time that miR-26a-5p expression was significantly down-regulated in human UTUC tissues compared with adjacent normal tissues. Moreover, the immunoexpression of miR-26a-5p decreased with higher T stage. The intensity score was lower in T3 tumors than T1 and T2 tumors (data not shown). In addition, www.nature.com/scientificreports/ the similar trends were found when compared to histological grade. This observation supports that high-grade tumor cells often constitutively express lower miR-26a-5p than low-grade tumor cells. Given that high histological grade is significantly related to aggressiveness and poor prognosis of tumor, our findings demonstrated that miR-26a-5p may play a tumor-suppressive role in the UTUC development. Consistent to our observation, the low miR-26a-5p expression has been earlier reported and identified as a poor prognostic marker in colorectal cancer due to poorer overall survival 30 . Therefore, it indicated a potential prognostic and therapeutic value of miR-26a-5p in UTUC. This is the first study to assay the miR-26a-5p expression in UTUC tissue samples. Moreover, our in vitro study showed that miR-26a-5p overexpression significantly suppressed the proliferation, migration and invasion of BFTC-909 cells. Furthermore, miR-26a-5p overexpression showed the inhibitory effect on EMT process which is associated with metastasis activity in UTUC cells. These results confirmed the inhibitory effect of miR-26a-5p on growth and metastasis in UTUC. Metastasis is one of the major causes of mortality in UC patients 31 . Therefore, the inhibition of metastasis is an important issue in UC research, including UTUC. There are studies showing that inactivation of NF-κB and the inhibition of the expression of MMP-9, ultimately suppressing invasion and metastasis 31,32 . In addition, it has been reported that high MMP-9 expression levels are associated with clinically aggressive tumors and worse prognosis [33][34][35] . In the current study, we had provided the evidence of miR-26a-5p inhibited the NF-κB and MMP-9 expression. Thus, miR-26a-5p might regulate UTUC via suppressing invasion and metastasis.
It is well known that a single miRNA can affect multiple targets via distinct mechanisms 36 . It had been reported the therapeutic usefulness of inhibition of Wnt/β-catenin signaling in cancers 27,37,38 . Moreover, studies had revealed that miR-26a inhibited cell proliferation, metastasis, EMT, β-catenin and enhanced apoptosis, E-cadherin via mediating WNT5A in cancers 6,27,39 . The current study demonstrated the function of miR-26a-5p in regulating UTUC might mediate through inhibition of WNT5A/β-catenin signaling. Recently, various miRNA replacement therapies are currently in clinical trial demonstrates the great potential of this approach to treat cancer 40 . Therefore, miR-26a-5p may be a novel therapeutic small molecule against UTUC.
In conclusion, the present study provides the evidence to support the anticancer properties of miR-26a-5p and act as tumor suppressors in UTUC cells. The major findings of the present study are summarized as a diagrammatic depiction (Fig. 7): miR-26a-5p down-regulation in UTUC, which may block WNT5A/β-catenin signaling and inhibit EMT process. Thus, replacement therapy with miR-26a-5p represents a promising novel therapeutic strategy against UTUC.

Materials and methods
Clinical specimens. All   www.nature.com/scientificreports/ Human UTUC tissue microarray (TMA). The human renal pelvis transitional cell carcinoma TMA containing 64 cases was purchased from US Biomax, Inc. (Kit no.KD642, Rockville, MD). The TMA slide was subjected to in situ hybridization (ISH) staining of miR-26a-5p, and quantified by pathologist according to the following rules. The labeling intensity was given a score from 1 to 4, corresponding to yellow, yellow-brown, brown and dark staining, respectively. The proportion of tumor cells with detectable cytoplasmic immunoreactivity for miR-26a-5p were also recorded using a 4-tier score, for 1 corresponding to < 30% tumor cells with positive staining, 2 for 30-60%, 3 for 60-80%, and 4 for > 80%. An expression index was defined as the product of these two scores mentioned above. Obviously, the index could range from 1 to 16, with 16 corresponding to > 80% tumor cells displaying dark (4) staining.
miR-26a-5p in situ hybridization. TMA slide was examined using in situ hybridization (ISH) staining. Tissue sections were stained with digoxigenin-labeled probe (sequence 5ʹ-AGC CTA TCC TGG ATT ACT TGAA-3ʹ) synthesized by BioTnA and complimentary to miR-26a-5p The expression level was measured by using a Biospot ISH detection kit (TASH01D, BioTnA, Kaohsiung, Taiwan). The signals were developed by DAB chromogen and documented under microscope as described 41 .
RNA isolation, reverse transcription and quantitative PCR (qPCR). Total RNA was extracted by using Trizol ® Reagent (Invitrogen, USA) according to the instruction manual as previously described 3 . To prepare a cDNA pool from each RNA sample, total RNA (10 ng) was reverse-transcribed using TaqMan  Cell proliferation assay. Cell proliferation assay was performed by using WST-1 assay kit (TaKaRa Cat. # MK400). All procedures were followed as previously described 3 .
Trans-well cell migration assay. Cell migration assay was performed using Trans-well chambers (Millicell, PIEP12R48). All procedures were followed as previously described 3 .
Immunofluorescent staining. BFTC909 cells were cultured in six-well glass slide chambers for 24 h and further transfected with miR-26a-5p mimics for 48 h. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 3% BSA for 30 min at room temperature. The fixed cells were then incubated with the primary antibodies against E-cadherin, vimentin, fibronectin, α-SMA, WNT5A, β-catenin, NF-κB, and MMP-9 at 4 °C overnight followed by visualization with Alexa Fluor 488-(green) or Alexa Fluor 595 (red)-conjugated secondary antibodies at room temperature for 1 h. Nuclei were counterstained with DAPI. The stained cells were mounted with a fluorescent mounting medium (Dako Cytomation) and observed under fluorescence microscopy (Olympus). The exposure gains and rates were consistent between samples. Fluorescent intensities were quantified on independent color channels by using Image J software (NIH, USA) 3 .

Statistical analysis.
All values in the figures were expressed as mean ± standard error of the mean and analyzed by unpaired t test. P value < 0.05 was considered to be statistically significant. www.nature.com/scientificreports/